Although surgical removal of malignant primary tumors is often successful, it remains difficult to halt metastatic spread or to predict which early stage cancers are potentially metastatic. Proteolytic enzymes are logical markers for the invasive potential of a tumor. Yet it is not clear how abnormal proteolytic activities are orchestrated to permit tumor spread. We propose that the step-wise sequence of changes in colorectal tumor progression provides a natural model system for studying the contribution of various proteinases to sequential events in tumor growth, local invasion and distant spread. Our data on stage- specific increases in cathepsin B and L expression with colorectal tumor progression raise new questions and provide a framework for our proposed research. One major hypothesis to be tested is that increased cathepsin B and L expression in early stage colorectal tumors represents the beginning of a tumor-specific proteolytic cascade with different proteinases activated in sequence at different points in the progression from "in situ" to metastatic carcinoma. Thus, we plan to characterize the expression of cathepsin H, cathepsin D and gelatinolytic type IV collagenases as potential partners with cathepsins B and L in a proteolytic cascade that may underlie colorectal tumor progression. Molecular changes associated with colorectal tumor progression, including ras oncogene activation and p53 mutations, may trigger or regulate critical steps in this proteolytic cascade. Thus, we also plan to determine whether ras activation in colorectal adenomas is closely correlated with the observed increase in cathepsin B mRNA or with appearance of the latent proprotein form of cathepsin B in adenomas. We will also test whether those earlier stage (Dukes' A and B) colorectal tumors that express elevated cathepsin B activity are the same subset of carcinomas that have both a ras and a p53 gene change. Defining these relationships between biochemical and molecular markers should make it possible to use altered proteinase expression to screen for an underlying molecular change. The ultimate goal of our research is to apply new information on proteinases to clinical diagnosis and prognosis. We have recently demonstrated that elevated cathepsin B expression in tumor extracts is also detectable by immunohistochemical staining of tumor cells on fixed, paraffin-embedded sections. Thus, using both enzyme activity measurements on tumor extracts and immunohistochemical staining of archival material, we propose to test whether, for different tumors within the same stage, cathepsin B is a useful, independent prognostic indicator of early, less aggressive colorectal carcinoma. As we characterize additional proteinases in colorectal tumors, we expect to identify similar but distinct stage-specific expression patterns that can be used to establish a set of markers that precisely define the potential for local invasion or distant metastasis of a given colorectal tumor.